1. Field of the Invention
The present invention is related to a total reflection illuminated sensor chip, which is employed in a method for detecting substances within samples by utilizing evanescent waves.
2. Description of the Related Art
Conventionally, detecting methods that utilize evanescent waves or surface plasmon induced by totally reflected illumination are being focused on, in biological measurements for detecting proteins, DNA, and the like. Surface plasmon are compression waves of free electrons which are generated by the free electrons vibrating as a group at the surfaces of metals. SPFS (Surface Plasmon Field enhanced fluorescence Spectroscopy) measurement is an example of a detecting method that utilizes the electric field enhancing effect of surface plasmon.
SPFS measurement is a method in which detection target substances are detected, by: generating evanescent waves on a metal film that functions as a detecting portion provided on a dielectric prism; exciting the detection target substances included in samples or fluorescent labels attached to the detection target substances by the evanescent waves; and detecting the fluorescence emitted by the detection target substance or the fluorescent labels. The evanescent waves are generated on the metal film, by causing a measuring light beam to be totally reflected at the interface between the dielectric prism and the metal film provided thereon. SPFS measurement is easy to execute, and is capable of measuring a plurality of samples simultaneously. Further, the electric field enhancing effect of surface plasmon, which is generated by the evanescent waves resonating with free electrons in the metal film, amplify the evanescent waves, and enables great fluorescent signals to be detected. Accordingly, SPFS measurement is widely used.
In the aforementioned SPFS measurement, measuring light beams are totally reflected at interfaces between dielectric prisms and a detecting portion. Therefore, dielectric prisms, on predetermined regions of which detecting portions are formed, are commonly employed as sensor chips (refer to Japanese Unexamined Patent Publication No. 9 (1997)-096605). Presently, dielectric prisms formed of plastic, which are less expensive and more easily molded than glass, are commonly employed.
The detection limits of SPFS measurement are determined by: (i) background light (leakage of light within a measuring apparatus); (ii) scattering of a measuring light beam; (iii) autofluorescence of an optical system due to the scattering of the measuring light beam; and (iv) background light formed by autofluorescence of a sensor chip, and not from fluorescent labels. The influence of (i) can be comparatively easily reduced by design of the apparatus. The influence of (ii) can also be easily reduced by providing a measuring light beam cutoff filter. The influence of (iv) is comparatively small, because excitation is performed via a metal film on the sensor chip.
However, in cases that the intended use of sensor chips is laboratory testing, it is not possible to expend much cost on the sensor chips. In this case, it is difficult to stably produce sensor chips that do not have burrs and ridged structures (polishing streaks) formed during the molding process. Therefore, it is difficult to avoid the influence of (iii) above. This is because portions of measuring light beams leak through polishing streaks on surfaces on which metal films are formed (metal film formation surfaces), resulting in increased noise.
In homogenous assay systems that take advantage of the characteristic of SPFS measurement, that only signals in the vicinity of a metal film are strongly excited, and perform fluorescence detection without performing cleansing to detect the amount of a detection target substance from the variance (rate) of fluorescent signals, another factor: (v) influence of fluorescence from floating labels due to scattering of a measuring light beam; is added, and becomes a grave problem.